牛分枝杆菌eis基因在耻垢分枝杆菌中的表达及其生物学活性鉴定

发布时间：2018-04-07 00:10

本文选题：eis基因　切入点：pMV　出处：《中国畜牧兽医》2017年07期

【摘要】：试验旨在构建牛分枝杆菌eis基因的穿梭表达载体,鉴定其在重组耻垢分枝杆菌中的生物学活性。采用PCR技术扩增并克隆牛分枝杆菌eis基因,构建大肠杆菌-分枝杆菌穿梭表达载体pMV261-Mbeis,经双酶切和测序鉴定其正确性,利用电穿孔法将重组质粒转化至耻垢分枝杆菌mc2155中,采用SDS-PAGE和Western blotting技术检测牛分枝杆菌eis基因在耻垢分枝杆菌中的表达,质谱鉴定目的蛋白氨基酸序列。研究结果表明,成功构建了牛分枝杆菌eis基因穿梭表达载体pMV261-Mbeis;生长曲线表明负载重组质粒不会影响耻垢分枝杆菌的体外生长;SDS-PAGE和Western blotting检测证实了牛分枝杆菌eis基因在耻垢分枝杆菌中可表达出分子质量约44ku的eis蛋白;质谱检测证明了该蛋白即为牛分枝杆菌eis蛋白。本研究构建的牛分枝杆菌eis基因穿梭表达质粒pMV261-Mbeis在耻垢分枝杆菌中具有生物学活性,为下一步研究表达产物eis蛋白的功能奠定了基础。
[Abstract]:In order to test the shuttle expression vector construction of EIS gene of Mycobacterium bovis, identification of the recombinant Mycobacterium smegmatis in biological activity. The amplification and cloning of EIS gene of Mycobacterium bovis by using PCR technology, construction of Escherichia coli Mycobacterium shuttle plasmid pMV261-Mbeis by double enzyme digestion and DNA sequencing. The recombinant plasmid was transformed into the correctness. To Mycobacterium smegmatis mc2155 by electroporation, to detect the expression of EIS gene of Mycobacterium bovis in Mycobacterium smegmatis by SDS-PAGE and Western Blotting Technology, mass spectrometry identification of target protein amino acid sequence. The results show that the successful construction of EIS gene of Mycobacterium bovis shuttle expression vector pMV261-Mbeis; growth curve showed that the recombinant load the plasmid does not affect the growth of Mycobacterium smegmatis in vitro; SDS-PAGE and Western blotting confirmed the detection of Mycobacterium bovis EIS gene in Mycobacterium smegmatis Bacteria can express a molecular mass of about 44ku EIS protein; mass spectrometry showed that the protein is the EIS protein of Mycobacterium bovis EIS gene of cattle. The constructed shuttle plasmid pMV261-Mbeis with biological activity in Mycobacterium smegmatis, which lays the foundation for further research of EIS protein products function.

【作者单位】：中国农业科学院北京畜牧兽医研究所;
【基金】：国家自然科学基金(31302130) 中国农业科学院科技创新工程(ASTIP-IAS-11)
【分类号】：S852.61

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